大鼠星状神经节快兴奋性突触后电位变化与三七总皂甙的影响

大鼠星状神经节快兴奋性突触后电位变化与三七总皂甙的影响 大鼠星状神经节快兴奋性突触后电位变化 与三七总皂甙的影响 中国组织工程研究与临床康复笫11卷笫25朋2007-06—24出版 JournalofClinicalRehabilitativeTissueEngineenngResearchJune24,2007.Vo1.11,No.25 EffectsofPanaxnotoginsengsaponinsonthe fast-excitatorypostsynapticpotentialin ratstellateganglion? ZhouYan,TianLei,MoNing 'Departmentof Pharmacology. GuangxiMedicaI Univemity.Nanning 530021.Guangxi ZhuangAutonomous Region,China; ZDepartmentof GastrointestinaI Surgery..Fi瞰 AffiIiatedHospitalof GuangxiMedicaI Univemity,Nanning 530027.Guangxi ZhuangAutonomous Region,China ZhouYan?.Studying fordootorate. Lecturer,Department ofPharmacoiogy, GuangxiMedicaI University,Nanning 530021.Guangxi ZhuangAutonomous Region,China ZhouytI@ yahoo.corn.cn Supportedby:the ScienUficResearch Founda~onof GuangxiEduca~on Bureau.No (20062659) 2OO6-2009. Received:20oB0B-28 Accepted:2007-03-14 (06-50-0-S454/YWY1 ZhouY,TianL.MoN. EffectsofPanax notoginsengsaponins onthafast.-excitato~ posts—ynapficpotent~l inratstellateganglion ZhongguoZuzhi GongchengYanjiuyu LinchuangKangfu 2007;11(25): 5044-5046(China) 【v『Ww.zglckfcom/ zglckf/ejoumal/ upfiles/07.25/ 25k-5044(ps).嗍 5044 Abstract BACKGROUND:Panaxnotoginsengsaponins(PNS)couldsignificantlyimprovethelearningandmemoryabilityofrats butitsinfluencetoperipheralnervoussystemstillneedsfurtherinvestigation. OB3EL-FIVE:ToobservetheeffectsofPNSonthefast-excitatorypostsynapticpotential(f-EPSP)instellateganglion (SG)ofrats. DESIGN:ObservationandcontrolledtdaI SE1-FING:PharmacologicalLaboratoryofGuangxiMedicalUniversity MATERIALS:TheexperimentwascarriedoutatthePharmacologicalLaboratoryoftheExperimentalCenterofGuangxi MedicalUniversityfr0mJanuary2005toFebruary2006.ThirtyhealthymaleSDratsofcleangradeand(220+_20)g, providedbytheExperimentalAnimaICenterofGuangxiMedicaIUniversity:SEN-7203digitalthreetrackstnpstimulator. microelectrodeamplifier(MEz8301,JapanNIHONKOHDENCOMPANY);glassmicroelectrodepuller,and microelectrodemanipulator.boththeproductsofNarishigeCompany,Japan;PNS,providedbyKunmingJacobson PharmaceuticaICo.,Ltd,andacetyIchloridechline(Ach),theproductofSigma,U.S.A. METHODS:Aftertheanimalswereexecutedacutely,theirchestwal1wasopenedtoIsolateSGrapidlyundermicroscope. whichwastransferredtotheperfusionchamber,andfixedwithwireneedlesafterpeelingtheconnectivetissue membrane.Thegangliawereperfusedcontinuouslywiththemixtureofvolumefraction0.95 O2and0.O5CO2plusKrebs solutionwithpH(7.4? O.05).Meanwhile,0.08-0.16g/LPNSwasemployedtopeseandcultureSG.(1)Theglass microelectrodefilledwith3mmol/LKC1wasusedtopuncturetheIsolatedSGandrecordtheamplitudeanddurationof depolarizingreactionofpostsynapticmembrane.? PNSwiththemaximumconcentrationof0.16g/L,whichcouldInhibit thef-EPSPs,wasperfusedtoobservetheeffectofPNSontheamplitudeanddurationofdepolarizingreactionof postsynapticmembraneInducedbyexogenousAch(1mmol/L.1minute).(3]PNSwiththemaximumconcentrationof O.16 口,L,whichcouldfnhibitthef-EPSPs,wasperfusedtoobservetheeffectofPNSonmembraneresistanceand membranepotentia1. MAINOUTCOMEMEASURES:(~Amplitudeofdepolarizingreactionofpostsynapticmembrane;?EffectofPNSonthe amplitudeanddurationofdepolarizingreactionofpostsynapticmembraneinducedbyexogenousAch,andmembrane resistanceandmembranepotentia1. RESULTS:ThirtyratswereinvolvedIntheresultanalysis.? PNSrangedO.O8toO.16g/Lcouldreversiblydepressthe f-EPSPsamplitudeof,orchangetheforwardactivepotentialintof-EPSP;thehighertheconcentrationofPNS,themore obvioustheInhibitionwas.ThedepressionappearedIn3-10minutesafterPNSperfusion.andtheeffectreachedthe peakat0.16g/L;f-EPSPwasdecreasedevidentlyin3to4minutes.TheInhibitionnearlyrecoveredtothecontrolleveI afterwashingthegangliawithKrebssolutionfor15t02Ominutes.(E仟 eclofPNSonexogenousACh.induced depolarization:TheamplitudeanddurationoftheAch-induceddepolarizationdidnotsignifi cantlychangebeforeand5 minutesafter0.16g/LPNSperfusion[before:(15.5?2.4)mV,(256.1? 21.5)seconds;after:(14.3?1.9)mV,(228.6+_24.5) seconds,P>0.051.(EffectsofPNSonmembranepotentialandmembraneresistance:The meanmembranepotentiaI andmembraneresistancewerenotsignificantlychangedafterPNSperfusionfbefore:一 (55.5?12.1)mV,(53.9?5.1)Mn: after:一(54.3?10.4)mV.(55.1?4.8)Mn,P>0.051. CONCLUSION:PNScouldreversiblydepressthefast-excitatorypostsynapticpotentialins tellateganglionofratsby presynapticmechanism. INTRoDUCTIoN Notoginseng,alsonamedpseudoginsengorradix notoginseng,thedryingrootofPanaxnotoginseng saponins(PNS),distributesmainlyinGugangxi,Yunnan andsoon.ItIssweetflavor,mildbitter,and warm-natured.andeffectiveInbloodcirculation amendment…,nervousexcitationInhibition,anti.anoxia[2i, immunoregulatory,anti—inflammation,anti-cadudtvand hepato-protection.PNSIstheimportantactive componentofradixnotoginseng:Rb1andRbthemain componentsofPNS,couldsignificantlyimprovethe IeamIngandmemoryabilitiesofratsi3】:moreover.PNS istheblockagentofsynaptosomecalciumchanneIof cerebraIcortex[41.However.therearenoreportsabout theInfluenceofPNSonperipheralnervoussystem. WIththeintracellularrecordingtechniques.theInfluence andactionmechanismofPNSonthesynaptic transmissionofste?ateganaIi0n(SG),0ne0fthe pe—pheraIsympathetIcgangI10n,andfast-excItat0ry p0stsynaptIcpotentiaI(f_EPSP),thec0mm0n postsynaptIcpotentiaI(PSP),areexp10rI?d. P.O.Box12o0,She吖ang11o0o4385oB3@}sina.oom州州.z9.踟 SSN1673—8225CN21—1539/R 周燕,等大鼠星状神经节快兴奋性突触后电位变化与三七总皂甙的影响 MATERlALSANDMETHoDS Materials TheexperimentwascarriedoutatthePharmacologicalLaboratory oftheExperimentalCenterofGuangxiMedicaIUniversityfr0m January2005toFebruary2006.ThidyhealthymaleSDratsof cleangradeand(220_+20)gprovidedbytheExperimentalAnimal CenterofGuangxiMedicaIUniversity『certificationnumber:SCXK (gui)2005-2006];SEN一7203digitalthreetrackstripstimulator, microelectrodeamplifier(MEZ8301,JapanNIHONKOHDEN COMPANY);glassmicroetectrodepuller,andmicroelectrode manipulator,theproductsofNarishigeCompany,Japan;IBBClamp dataacquisitionsoftware,theproductofYiboCompany,Huazhong UniversityofScienceandTechnology. Drugsandagents:PNS,providedbyKunminqJacobson PharmaceuticaICo.,Ltd:acetyIch10ridechline(Ach),theproductof Sigma.U.S.A.OtheragentswereanalyticalpuremadeinChina. Methods Preparationandperfusionofganglionsamples Aftertheanimalsweresacrificedacutely,thechestwal1was opened,andSGtogetherwiththeirpreganglionicnervetrunkswere isolatedrapidly,thentransferredtotheperfusionchamber.and fixedwithwireneedlesafterpeelingtheconnectivetissue membrane.Thegangliawereperfusedcontinuouslywiththe mixtureofvolumefraction0.95O,and0.O5CO,plusKrebs solution[withpH=(7.4+0.05).whichwascomposedof117mmol/L NaCI,47mmol/LKCI,2.5mmol/LCaCI2,1.2mmol/LMgCI,, 25mmol/LNaHCO3,1.2mmol/LNaH2PO4,and11.5mmol/L Glucose.Meanwhile.0.O8一O.16g/LPNSwasemployedtoperfuse andcultureSGforfollowingexperiment. Intracellularrecordingandindexdetection Theglassmicroelectrodewith3mmol/LKCland30—40Mntop impedancewasfixedtothemanipulatorforcelIpuncture. SEN一7203digitalthreetrackstripstimulatorandstimulusisolator suppliedsquarewavecurrentordirectcurrent,whichwasperfused intotransmembranecurrentthroughglassmicroelectrode. BioelectricaIpotentialsignalswereamplifiedbyamicroelectrode amplifier(MEZ8301),andinputonanoscilloscope,aswelIasa penrecorder,andstoredinfilesonacomputeratthesametime Whentheglassmicroelectrodewasinsertedintothecells.the electronbeamonoscilloscopeabruptlydownwarddeflected,and thedeflectionvaluesrepresentedthecelImembranepotentiaI values:theconstanthyperp0Ianzinqcurrentpulseof0.2—0.5nA couldinduceelectrotonicpontentialofcellularmembrane. thenthe membraneresistancewascalculatedbyOhm.sIaw(尺=V/I) accordingtopreviousvalues.Achdirectpeffusionof1mmol/Lfor 1minutecouldbeusedtorecordtheamplitudeanddurationof depolarizingreactionofpostsynapticmembrane.Thef-EPSPswere evokedbystimulatingthepreganglionicnevetrunkwithsinglepulse atO.2一O.5Hz.O.5—1.0msand2—1OV【0-. Statisticalanalysis ThedatawereexpressedasMean+SDandtwo—tailedpairedftest wasusedforstatisticalcomparisonbeforeandafteradministration. RESULTS Animalquantitativeanalysis Thirtyratswereallinvolvedintheresultanalysiswithoutanyloss PNSranged0.08t0016g/Lcouldreversiblydepressthe amplitudeoff-EPSPs(Table1)orchangetheforwardactive potentialintof-EPSP;thehighertheconcentrationofPNS,the moreobvioustheinhibitionwas.Thedepressionappearedin3—10 minutesafterPNSpeffusion,andtheeffectreachedthepeakat 0.16g/L.f-EPSPwasdecreasedevidentlyin3to4minutes. f-EPSPnearlyrecoveredtothecontroIIeveIafterwashingthe gangliawithKrebssolutionfor15102Ominutes(Figure1). PNS:Panaxnotoginsengsaponins;f-EPSP:fast—excitatorypostsynaptic potentials;.P<0.01."一"representsthedecreasebasedonoriginallevels a:O5Hzsingle pulsestimulationto sympatheticnerve trunkcouldinduce f-EPSP b:Threeminutesafter O16g/LPNSpedusion, f.EPSPwasdecmased. and6minuteslater. f-EPSPwasdecreased by7O% c:Afterwashedwith Kreb'ssolutionfor15 minutes,amplitudeof f-EPSPlevels recoveredtothe controllevels V PNs=Panaxnotoginsengsaponins;f-EPSP:fast—excitatorypostsynaptic Potentials Figure1InhibitoryeffectofPanaxnotoginsengsaponinson fast-excitatorypostsynapticpotentialsinstellateganglionofrats EffectofPNSonexogenousACh-induceddepoIarization ThechangesinAch—induceddepolarizationreflectedthesensitivity ofnicotinicreceptorstoAchThegangliaswerepe~used continuouslywithPNSatthemaximumconcentration(0.16g/L), whichcouldinhibitf-EPSP,toobservetheeffectofPNSonthe remarkabledepolarizationinducedbyexogenousAch(1mmol/L. 1minute).Inthe7neuronstest.theamplitudeanddurationofthe Ach-induceddepolarizationdidnotsignificantlychangebeforeand5 minutesafter0.16g/LPNSpe~usion[before:(15.5?2.4)mV, (256.1+21.5)seconds;after:(14.3+1.9)mV,(228.6+24.5) seconds】,respectively. EffectsofPNSonmembranepotentialandmembrane resistance Thegangliaswerepe~usedcontinuouslywithPNSatthemaximum concentration0.16g/L,whichcouldinhibitf-EPSP.toobservethe effectofPNSonmembranequailty.In15neuronstest. mean membranepotentialandmembraneresistancewerenotsignificantly changedafterPNSpe~usionbasedontheelectrotonicpotentiaI changesinducedbyhyperpolarizingcurrentpulse[before:一(55.5? 12.1)mV,(53.9?5.1)Mn:after:一(543?10.4)mV,(551?4.8)Mn】. DlSCUSSloN EffectsofPNSonsynaptictransmissioninSGshowthat010一 EffectofPNSonf-EPSP0.16g/LPNShasthereversiblyinhibitoryeffectonf-EPSPin Theresultscamefr0mthe46impaledwellSGneuronsandstablysympatheticganglia.andthe inhibitoryeffectisenhancedfollowing reco~edwithmeanmembranepotentialof(5130? 11.45)mV.theincreaseofconcentrationofPNS.PNSreversiblydepressed 沈阳1200邮政信箱110004kf23385083@sinacomwwwzglckfcom5045 一 \ 八二- ISSN1673.8225CN21-15391R 周燕,等大鼠星状神经节快兴奋性突触后电位变化与三七总皂甙的影响?c? 7kf23385083@sina.com theamplitudeoff-EPSP,butdidnotaffecttheexogenous ACh—induceddepolarization,membranepotentialandmembrane resistance. f-EPSPisgeneratedbythepostsynapticnicotinicreceptorsacting withtheAchreleasedfr0mpresynapticneverterminaIafter preganglionicsimulations,inwhichthepresynapticmembrane nerveisdepolarizedaftertheimpulsereachesthenervetermina1. andthevoltagedependentCachanneIisopen.andIeadstothe infiuxofC:a2【8一i.InflowCapromotesthevesicletofusewith presynapticmembrane,toreleasethetransmitterAch,andfinally Ieadtothegenerationoff-EPSP.Thus.theinhibitoryeffectsof PNSonf-EPSParedisplayedbytwoprobablemechanisms: Presynapticmechanism.inwhichPNSdecreasesAChreleasefr0m presynapticneverterminal;?Postsynapticmechanism,inwhich PNSdecreasesthepostsynapticsensibilitytoAchorhassome directactiononpostsynapticmembrane. Thefindingsshowthat(1)Attheconcentrationthatcoulddepress f-EPSP.PNSdoesnotalterthemembranepotentialand membraneresistancesignificantly,indicatingPNShasnodirect effectonpostsynapticmembrane;?AlthoughPNScoulddepress f-EPSP.itcouldnotsignificantlychangethemembrane depolarizationinducedbyexogenousAch.indicatingtheinhibition ofPNStof-EPSPisnotinducedbythedirectdecreaseinthe postsynapticsensibilitytoAch,butbychangingAchrelease,itisa kindofpresynapticinhibition.AsthereleaseofAchisclosely relatedwithinfiowofCa【一i,moreover.PNShasbeenfoundthe blockagentofsynaptosomecalciumchanneIofcerebraIcortex[41 inthenervoussystemresearches,andPNSmonomerRbl displaysaninhibitioneffectonLtypevoltage—dependentcalcium channeIinadose-dependentmanneri10】incardiovascularsystem researches,itisablockagentofcalciumchanneI.Therefore.the inhibitioneffectofPNSonf-EPSPinSGisprobablyrelatedwith theinhibitiononCa"inflowonthebiologicalmembraneof peripheralsympatheticganglion,asthedecreaseinCainflow couldreducethereleaseofAch.andIeadtothedecreaseinthe amplitudeoff-EPSP.whiletheinhibitionofPNSonCachanneI andtheprecisemechanismstilIneedsfurtherstudy. 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NeurosciLett1986:64:263—268 DunNJ.JiangZG.MoNLong一【ermfacilitationofpeptidegic transmissionbycatecholaminesinguineapiginferiormessntedc ganglia.JPhysiol1984357(11:37—50 LlinasR.SngimoriM.SilverRB.Microdomainsofhighcalcium concentrationinapresynaptictermina1.Science1992;256(11:677—679 SmithSJ,BuchananJ,OssssLR,eta1.Thespatialdistributionof calciumsignalsinsquidpresynaptic.J.Physiol1993;472(1):573—593 ZhangB,JinSA,KuangX.eta1.EffectsofRb,oncalciumchannelin guineapigventricularmyocytes.ZhongguoYaolixueTongbao1998;14 (1):33—5 大鼠星状神经节快兴奋性突触后电位变化与 三七总皂甙的影响? 周燕',田磊,莫宁' '广西医科大学药理学教研室,广西壮族自治区南宁市530021;广西 医科大学第一附属医院胃肠外科,广西壮族自治区南宁市530027 周燕?,女,1975年生,湖北省武汉市人,汉族,2003年广西医科大学 毕业,在读博士,讲师,主要从事神经药理学的研究. 广西区教育厅科研项目资助【(20062659)2006—2009]' 摘要 背景:有研究显示,三七总皂甙能明显增强大鼠的学习与记忆能力,但其 对外周神经系统的作用仍需进一步观察. 目的:观察三七总皂甙对大鼠星状神经节快兴奋性突触后电位的作用. :观察对照实验. 单位:广西医科大学药理学教研室. 材料:实验于2005—01/2006—02在广西医科大学实验中心药理实验室 完成.选用30只健康雄性清洁级SD大鼠,体质量(220+20)g,由广西 医科大学实验动物中心提供.SEN一7203数字式三通道刺激器, MEZ8301型微电极放大器为日本NIHONKOHDEN公司产品,玻璃微 电极拉制仪,微电极操纵仪均为日本Narishige公司产品.三七总皂甙 (为云南昆明雅阁臣药业公司产品批号020802)氯化乙酰胆碱(Ach)为 美国Sigma公司产品. 方法:大鼠麻醉后迅速处死,打开胸壁,在显微镜下将星状神经节离体 并将标本迅速移至灌流浴槽,剥去神经节外层结缔组织膜,用金属细 针固定其边缘.用含体积分数0.95O2与体积分数0.05CO2混合气体 和pH为(7.4~005)的Kreb's持续,恒温(34.0~0.5?)灌流神经节, 同时采用质量浓度范围0.08~0.16g/L三七总皂甙对神经节进行灌流 和培养.用内充3mmol/LKCI的玻璃微电极穿刺离体星状神经节 神经元,细胞内突触后膜除极反应的幅度.?选用三七总皂甙可 逆性抑制快兴奋性突触后电位的最高浓度O.16g/L直接灌流.观察三 七总皂甙对外源性Ach(1mmol/L,1min)引起的突触后膜除极反应幅 度和时程的影响.?选用三七总皂甙可逆性抑制快兴奋性突触后电位 的最高浓度O.16g/L直接灌流,观察三七总皂甙对膜性质(膜电阻和膜 电位)的影响. 主要观察指标:细胞内突触后膜除极反应的幅度.?最高浓度0.16g/L 三七总皂甙对外源性Ach引起的突触后膜除极反应幅度和时程及对膜 性质(膜电阻和膜电位)的影响. 结果:纳人大鼠30只均进人结果分析.?三七总皂甙对快兴奋性突触 后电位的影响:三七总